Infection, Genetics and Evolution 40 (2016) 186–191

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Infection, Genetics and Evolution

j ourna l homepage: www.e lsev ie r .com/ locate /meeg id

Comparative molecular epidemiology of two closely relatedcoronaviruses, bovine coronavirus (BCoV) and human coronavirus OC43(HCoV-OC43), reveals a different evolutionary pattern

Nathalie KIN a,b,⁎, Fabien Miszczak a,b,c, Laure Diancourt d, Valérie Caro d, François Moutou e,Astrid Vabret a,b,c, Meriadeg Ar Gouilh a,b,d

a Normandie Université, 14032 Caen, Franceb Université de Caen, Unité de Recherche Risques Microbiens (U2RM), F-14000 Caen, Francec Laboratoire de Virologie, Centre Hospitalo-Universitaire de Caen, F-14033 Caen, Franced Institut Pasteur, Environment and Infectious Risks Research and Expertise Unit, F-75015 Paris, Francee ANSES, Boulogne Billancourt, F-92012, France

⁎ Corresponding author at: Normandie Université, 1403E-mail address: nathalie.kin@unicaen.fr (N. KIN).

http://dx.doi.org/10.1016/j.meegid.2016.03.0061567-1348/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 September 2015Received in revised form 4 March 2016Accepted 6 March 2016Available online 9 March 2016

Bovine coronaviruses (BCoVs) are widespread around the world and cause enteric or respiratory infectionsamong cattle. The current study includes 13 samples fromBCoVs collected inNormandy during an11-year period(from 2003 to 2014), 16 FrenchHCoV-OC43s, and 113 BCoVs or BCoVs-like sequence data derived frompartial orcomplete genome sequences available on GenBank. According to a genotyping method developed previously forHCoV-OC43, BCoVs and BCoVs-like are distributed on three main sub-clusters named C1, C2, and C3. Sub-clusterC1 includes BCoVs and BCoVs-like fromAmerica andAsia. Sub-cluster C2 includes BCoVs from Europe. Sub-clusterC3 includes prototype, vaccine, or attenuated BCoV strains. The phylogenetic analyses revealed themonophyleticstatus of the BCoVs from France reported here for the first time. Moreover, BCoV exhibits a relative geneticstability when compared to HCoV-OC43we previously described from the same region. The numerous recombi-nation detected betweenHCoV-OC43weremuch less frequent for BCoV. The analysis points thus to the influenceof different evolutive constraints in these two close groups.

© 2016 Elsevier B.V. All rights reserved.

Keywords:GenotypingBovine coronavirusHuman coronavirus OC43EvolutionEpidemiologyRecombinationRespiratory infection

1. Introduction

Coronaviruses are the largest enveloped single-strand RNA viruses,ranging from 26 to 31 kilobases in size. They belong to the Coronaviridaefamily in theNidovirales order, also including the families of Arteriviridae,Roniviridae, and the last described Mesoniviridae (Lauber et al., 2012;Gorbalenya et al., 2006).

Coronaviruses are divided into four genera named Alpha-, Beta-,Gamma- and Deltacoronavirus based on phylogenetic distance of highlyconserved domains (Adams and Carstens, 2012; Woo et al., 2012).Betacoronavirus genus is divided into the four clades A to D.Coronaviruses infect a wide range of avian or mammalian species, andare responsible for enteric or respiratory infections (Woo et al., 2009).Interest in coronaviruses increased by the emergence of two humancoronaviruses, Severe acute respiratory syndrome-related coronavirus(SARS-CoV) and Middle-East respiratory syndrome coronavirus(MERS-CoV) in 2002 and 2012, respectively. Both these coronavirusesbelong to the Betacoronavirus genus, clades B and C, respectively, and

2, Caen, France.

cause severe respiratory diseases in humans. SARS-CoV emerged inhuman in 2002–2003, causing an outbreak with more than 8000 caseswith a fatality rate of nearly 10% (Peiris et al., 2004).MERS-CoV emergedin 2012 in the Middle-East (Zaki et al., 2012). To date, 1413 laboratorycases of MERS-CoV infection have been confirmed, including 502 fatalcases, reported by the World Health Organization (WHO) (InstitutNational de Veille Sanitaire (INVS), 2015). Both SARS-CoV and MERS-CoV have a zoonotic origin, with an animal reservoir in close contactwith the human population. The zoonotic potential of coronavirusesimplies the need for surveillance of coronaviruses associated withdomestic animals in close contact with human population.

Bovine coronavirus (BCoV) belongs to the Betacoronavirus genusclade A. This clade also contains other closely related coronavirusesknown as Bovine-like coronavirus (BCoV-like), originating from captivewild ruminants such as waterbuck (Kobus ellypsiprimus), sambar deer(Cervus unicolor), white-tailed deer (Odocoileus virginianus), elk (Cervuselephus), giraffe (Giraffa camelopardalis), and sable antelope (Hipotragusniger) (Alekseev et al., 2008). These coronaviruses were antigenically,biologically, and genetically highly related to BCoV (Alekseev et al.,2008; Tsunemitsu et al., 1995; Majhdi et al., 1997; Hasoksuz et al.,2008). BCoVs-like were also detected from camelids such as alpaca

187N. KIN et al. / Infection, Genetics and Evolution 40 (2016) 186–191

(Vicugna pacos), llama (Lama glama), and dromedary camel (Cameliusdromedarius). Particularly, a BCoV-like, named Dromedary camel corona-virus (DcCoV), was detected from dromedary camel feces in the UnitedArab Emirates in 2013, during an epidemiological investigation aimingto identify the animal reservoir of MERS-CoV (Cebra et al., 2003; Wooet al., 2014). BCoVs-like are globally responsible for enteric infection.However, in 2002 a BCoV-like responsible for respiratory infection wasdetected from a dog in United Kingdom (Erles et al., 2003). To date,only 15 complete genomes of BCoVs, including 13 from USA, one fromCanada, and one from South Korea are available on GenBank. Moreover,16 BCoVs-like complete genomes, including 12 from captive ruminants,one from a dog, and three from dromedary camels are available onGenBank (consulted on September 8th 2015). Much earlier, in 1988, acoronavirus closely related to BCoV was isolated from the feces of adiarrheic child in Germany. This coronavirus, tentatively named at firstHuman enteric coronavirus (HEC 4408), revealed itself to be closer toBCoVs than to BCoV-Like and to other HCoVs indeed. The phylogeneticposition of this virus and the context of this case suggests the infectionof the child by a cattle virus, consequently to a species barrier-crossing(Zhang et al., 1994).

BCoV is closely related to the Human coronavirus OC43 (HCoV-OC43), that was first isolated in 1967 (McIntosh et al., 1967). Moreprecisely, BCoV and HCoV-OC43 share a global nucleotide identityof 96%. The genetic comparison of BCoV and HCoV-OC43 revealsthat HCoV-OC43 results from a zoonotic transmission from bovine tohuman. Using a molecular clock analysis, Vijgen et al. estimatedthe tMRCA of these viruses around 1890 (Vijgen et al., 2005). The intra-specific variability of the HCoV-OC43 revealed the co-circulation ofnumerous recombinant variants (Kin et al., 2015). Given the similarityof HCoV-OC43 with BCoV, we were interested in testing the geneticvariability of BCoV using the same methods. Thus, the objective of thecurrent study was to investigate the intraspecific genetic variability ofBCoV collected in Normandy from 2003 to 2014 and to test whetherrecombination remain a major mechanism of variability in BCoVs asseen for his sister clade HCoV-OC43.

Fig. 1. Location of the livestockwhere nine of our sampleswere collected. Thenameof samplewThe nine corresponding farms were named F1 to F9.

2. Materials and methods

2.1. Samples and positive control

Thirteen field samples positive for BCoV and one cell-culture BCoVsupernatant were included in this study. The 13 field samples wereprovided to us by laboratories of Normandy specializing in veterinarydiagnostics. These samples include 12 diarrheic fecal samples and onenasal aspirate. The cattle herd of origin of nine samples are known pre-cisely and depicted in Fig. 1. The cell-culture supernatant is derived froma fecal sample propagated onHRT-18 cells in our laboratory since 1990s.We used this BCoV as positive control. Age, type of samples and countiesof origin of specimens, when available, are indicated in Table 1.

2.2. Sample preparation, nucleic-acid extraction, and real-time RT-PCR

Fecal samples were resuspended (10% suspension in sterile PBSBuffer) and centrifuged at 3000 rpm for 10 min. Total nucleic acids of200 μl of fecal suspension, nasal aspirate, and culture supernatantwere manually extracted using High Pure RNA Isolation Kit (Roche,Basel, Sweden) following the manufacturer's instructions. The determi-nation of the cycle threshold (Ct)was performed using an in-house real-time RT-PCR, targeting a 128 nucleotide fragment of the BCoV M gene,for which the original primers and probe can be found in Table S1(supplementary material). RT-PCR reactions were performed usingOne StepRT-PCR System (Qiagen,Holden,Germany). Reactionmixtureswere performed in a final volume of 25 μl, composed of 5 μl of 5× reac-tion buffer, 1 μl of a dNTP solution (0.4 mM each), 1 μl of Enzyme mix(Reverse transcriptase and Taq polymerase), and 12 μl of RNA freewater, provided in the kit. We added 1.5 μl of MgSO4, 0.4 μM of eachBCoV-M550F and BCoV-M650R primers, 0.2 μM of BCoV-M600 probe,and 2.5 μl of RNA template. Thermal cycling involved 50 °C for 30 min,followed by 95 °C for 15 min and then 45 of 95 °C for 30 s and 60 °Cfor 1 min.

as indicated above each farmof origin as follows: Caen/year of sampling/specimennumber.

Table 1Features of the 14 BCoV samples used in this study. amo., month; d., days. bna, notavailable.

Specimen Age at timesamplinga,b

Type of sample Department oforiginb

Caen/2005/01 na Respiratory CalvadosCaen/2005/02 na Enteric CalvadosCaen/unknown/03⁎ na Culture supernatant (HRT18) naCaen/2008/04 b1 mo. Enteric MancheCaen/2003/05 na Enteric CalvadosCaen/2010/06 5 d. Enteric MancheCaen/2012/07 b1 mo. Enteric MancheCaen/2013/08 15 d. Enteric MancheCaen/2013/09 b1 mo. Enteric MancheCaen/2013/10 b1 mo. Enteric MancheCaen/2013/11 b1 mo. Enteric MancheCaen/2014/12 b1 mo. Enteric MancheCaen/2014/13 b1 mo. Enteric MancheCaen/2004/14 7 d. Enteric Calvados

⁎ Propagated on cell culture since 1990s.

188 N. KIN et al. / Infection, Genetics and Evolution 40 (2016) 186–191

2.3. Sequencing of the three complete genes nsp12, S, and N and genotypingmethod

The sequences of BCoVs were named according to the followingnomenclature: Virus name/FRA-EPI/location of sampling/year ofsampling/specimen number. FRA corresponds to the acronym ofFrance, EPI is an abbreviation for the EPICOREM consortium. In thisstudy, the term “molecular isolate” was used for sequences of BCoVamplified directly from samples.

To determine the genomic sequence of nsp12, S, and, N genes, a setof 17 overlapping RT-PCR products, from 553 to 1260 nucleotides, weregenerated using a total of 42 primers (Lau et al., 2011; Hasoksuz et al.,2002; Martínez et al., 2012). RT-PCR reactions were performed usingOne Step RT-PCR System (Qiagen, Holden, Germany), from 2.5 μl ofnucleic acid in a final volume of 25 μl, according the manufacturer'sinstructions. Briefly, reaction mixtures were composed of 5 μl of 5×reaction buffer, 1 μl of a dNTP solution (0.4 mM each), 1 μl of Enzymemix (Reverse transcriptase and Taq polymerase), and 13.5 μl of RNAfree water, 0.4 μM of forward and reverse primers (Table S1, supple-mentarymaterial), and 2.5 μl of RNA template. Thermal cycling involved50 °C for 30 min, followed by 95 °C for 15 min and then 45 of 95 °C for30 s, 56 °C to 60 °C (depending of primers used), and 72 °C for 1 min.

The RT-PCR products encompassed all three complete genes(Table S1, supplementarymaterial). All the primers were tested in silicoand in vitro with our control BCoV strain to test their efficacy and theprimer abilities to provide us with quality sequencing products.

The bidirectional sequencing was performed at the Laboratory forUrgent Response to Biological Threats, Environment and InfectiousRisks Unit, Institut Pasteur (Paris, France), following their sequencingprotocol. The analysis of sequencing products was performed by thecapillary electrophoresis 3730XL (Life Technologies, Carlsbad, CA, US).

2.4. Bioinformatic analysis

Sequences were assembled in contigs corresponding to the entirensp12, S, or N genes with CodonCode Aligner Software, version 5.0.1(CodonCode corporation, Centerville, MA, USA). Multiple sequencealignments were performed using Muscle algorithm, implemented inMEGA software, version 6.06 (http://www.megasoftware.net). ThirteenBCoV sequences from the USA, available in Genbank, were added to thealignment (Tables S2 and S3, supplementary material) (Chouljenkoet al., 2001; Zhang et al., 2007; Mebus et al., 1973). Two additionalprototype BCoV sequences from Japan and Canada, named Kakegawaand Quebec respectively, were also added (Gélinas et al., 2001; Yooand Pei, 2001). Sixteen sequences from bovine-like coronaviruseswere added as well, corresponding to coronaviruses collected from

sable antelope, white-tailed deer, sambar deer, waterbuck, giraffe,alpaca, dog, and dromedary camel (Alekseev et al., 2008; Cebra et al.,2003; Woo et al., 2014; Erles et al., 2003; Hasoksuz et al., 2007). Finally,the sequence of HEC 4408 published by Zhang et al. and the 16sequences of HCoV-OC43 previously published by our team wereadded (Zhang et al., 1994; Kin et al., 2015). The accession numberscorresponding to these sequences are given in Tables S2 and S3 (supple-mentarymaterial). Note that there are no complete genomes of BCoV orBCoV-like isolates from Europe.

Maximum likelihood phylogenetic trees were constructed using theTamura-Nei's (TN93) substitution model, according to Akaike Informa-tion Criterion (AIC) and Bayesian Information Criterion (BIC) valuesevaluated by a model testing (Aldrich, 1997; Tamura and Nei, 1993). Abootstrap test including 1000 replicates was performed for each tree(Felsenstein, 1985). All of these programs are implemented in MEGA6software (Tamura et al., 2013). The topologies of trees were alsodouble-checked using a neighbor-joining method (substitution modelTN93), with a bootstrap test including 1000 replicates (Felsenstein,1985; Saitou and Nei, 1987). The topologies of these trees constitutethe basis of our genotyping methodology. The genotype of each BCoVor BCoV-like is defined based on the sub-cluster in which the threensp12, S and N sequences of a same BCoV (or BCoV-like) is located oneach tree. Finally, the genotypename is constructed by the juxtapositionof the sub-cluster names, C1, C2 or C3, on nsp12, S, and N treerespectively.

An additional maximum likelihood tree based on 143 completesequences of S gene of BCoV, BCoV-like, and HCoV-OC43 from USA,Brazil, South Korea, China, Italy, Swedish, Denmark and Francewas con-structed. To this end, 81S gene sequences from BCoVs and BCoVs-likewere added to the previous S gene alignment (Martínez et al., 2012;Beaudeau et al., 2010; Decaro et al., 2009a; Jeong et al., 2005; Zhanget al., 1991; Park et al., 2007; Chung et al., 2011; Park et al., 2006). Theaccession numbers corresponding to these sequences are provided inTables S2 and S3 (supplementary material). Phylogenetic trees wereconstructed using the maximum likelihood method with the substitu-tion model TN93, implemented in MEGA6 (Aldrich, 1997; Tamura andNei, 1993; Tamura et al., 2013). The topology of the tree was checkedusing a neighbor-joining (substitution model TN93) method, with abootstrap test including 1000 replicates (Felsenstein, 1985).

3. Results

Thephylogenetic analysis presented in Fig. 2 shows thensp12, S, andN trees obtained by a maximum likelihood method. Two clusters, onegrouping BCoVs and BCoVs-like and the other one containing HCoV-OC43s, are observed in the nsp12 and N trees. DcCoVs differentiates asa potential third cluster on the S tree. The BCoV cluster of the nsp12, S,and N maximum likelihood trees is divided into three sub-clusters.These sub-clusters are globally comprised of by the samemolecular iso-lates for nsp12, S, and N trees. Nsp12, S, and N produced comparabletree topologies and therefore a globally congruent phylogenetic signalthat does not suggest BCoV recombination events, contrary to HCoV-OC43 during the same period and in the same geographic area (Kinet al., 2015). The positions of BCoV and BCoV-like sequences on eachtree are summarized in Table S2 (supplementary material). Accordingto nsp12, S, and N trees, one sub-cluster named C1 contains BCoVs andBCoVs-like from the USA, constituting the genotype C1C1C1. A secondsub-cluster, named C2, groups on each trees all the BCoV sequencesgenerated in this study defining the genotype C2C2C2. Finally, the lastsub-cluster, named C3, contains the prototype strains Kakegawa,Mebus, Quebec, and CrCoV on all trees, constituting the genotypesC3C3C3. Two exceptions are observed. First, the three DcCoVs are includ-ed on sub-cluster C2 on nsp12 and N tree, but are grouped in a potentialoutlier position from BCoV and HCoV-OC43 clusters, constituting agenotype named C2COUTLIERC2. The second exception is the HEC4408 that is located on sub-cluster C3 on nsp12 and N trees, and in

Fig. 2. Phylogenetic analysis of the complete nsp12, S, andN genes of 29 BCoVs, 16 HCoV-OC43s, 17 bovine-like CoVs. The phylogenetic treeswere constructed by themaximum likelihoodmethod. Bootstrap values were calculated from 1000 replicates. Bootstrap values over 70% are shown. The evolutionary distances were computed using the Tamura Nei model and unitsare the number of base substitutions per site.

189N. KIN et al. / Infection, Genetics and Evolution 40 (2016) 186–191

sub-clusters C2 on S tree, constituting the genotype C3C2C3. This analysisreveals a potential geographical distribution with a segregation of BCoVand BCoV-like sequences fromUS on onehand, and BCoV fromFrance inanother hand. An additional S tree, depicted in Fig. S4 (supplementarymaterial), includes additional BCoV and BCoV-like sequences from theUSA, Brazil, South Korea, China, Sweden, Denmark, and Italy. The topol-ogy of this additional S tree is concordant with the S tree presented inFig. 2. The same tree clusters (BCoV and BCoV-like, HCoV-OC43, andDcCoV) were observed. In the same way, the three sub-clusters C1, C2,and C3 were observed inside to the BCoV cluster. The additionalsequences include in this most detailed S tree allowed us to deeplycharacterize the geographic distribution of BCoV and BCoV-like. Indeed,in addition to USA sequences, the sub-cluser C1 contains BCoVs andBCoVs-like from both America and Asia continent, more preciselyfrom China, South Korea, and Brazil. The sub-cluster C2 contains in addi-tion to the 14 French BCoVs, 35 additional BCoVs from other Europeancountries such as Sweden, Denmark, and Italy (cf. Tables S2 and S3 insupplementary material).

4. Discussion

The world population of cattle is estimated at 1.43 billion (Robinsonet al., 2014). In France, the cattle population extends to around 19million among which 1.9 million constitute the livestock of Lower

Normandy, where the present study emerges from. BCoV infectionsare not listed as “monitored transmittable veterinary infections”. There-fore, no public data measuring the impact of this infection on livestockcattle, national or international, are available despite BCoV being anubiquitous virus. Partial genomic sequences available in GenBankcome from 17 countries, unequally distributed on five continents.Fifteen complete BCoV genomes from North America and South Korea(14 and 1, respectively) are currently available (according to GenBank,consulted on September 8th, 2015) but no BCoV sequence fromFrance was available before the present study. Among the few availableepidemiological studies, Beaudeau et al. showed that in Sweden, theseroprevalence of BCoV is ranging from 43 to 65% in cattle less than12months, and observed a winter circulation in the temperate zones ofthe Northern hemisphere (Beaudeau et al., 2010). This epidemiologyshares common characteristics with that of the HCoV-OC43 infectionin humans, namely a primary infection in young patients in winter.BCoV and HCoV-OC43 belong to the clade A of Betacoronavirus genussharing a global similarity of 96% on a nucleotide level. The divergencebetween HCoV-OC43 and BCoV is estimated at the end of the 19th cen-tury, thus contemporary with a species barrier crossing from cattle tohumans. Six different genotypes have been identified in France amongthe 15 HCoV-OC43 circulating strains (including numerous recombi-nant variants) in respiratory samples taken over a 13-year period (Kinet al., 2015). This evolutive pattern (deeply affected by recombination)

190 N. KIN et al. / Infection, Genetics and Evolution 40 (2016) 186–191

has been commonly described for other coronaviruses (Infectionbronchitis virus, Feline coronavirus, Canine coronavirus) and proves, inthese contexts, to be a factor that favors the potential emergence ofnew variants (Cavanagh et al., 1992; Herrewegh et al., 1998; Decaroet al., 2009b). There is no published data for the potential localizationof recombination hot spot(s) on the BCoV genome. Given the genetic re-latedness between HCoV-OC43 and BCoV, we propose the hypothesisthat these potential hot spots could be located in the same regions forthese two viruses. Extensive sequencing of complete BCoV genomeswould ascertain the existence of these hot spots, but was not performedin this study.

The addition of BCoV sequences in France into the phylogeneticanalysis does not modify global phylogeny since HCoV-OC43 andBCoV remain monophyletic in our analysis. This holds true even whenthe prototype strains (propagated in cellular culture) are included inour analysis (data not shown). In this light, BCoVs from France aregrouped in a non-ambiguous way with the other BCoVs but remainthemselves monophyletic. Base on complete or partial genome phylo-genetic trees, BCoVs-like form a sister group to BCoVs and includeBCoVs from various animal origins (Alekseev et al., 2008; Woo et al.,2014).

The topological analysis of the three phylogenetic trees obtained,nsp12, S, and N, shows the existence of two clusters, one includingHCoV-OC43 and the other including both BCoV and BCoV-like, respec-tively. The BCoV cluster is divided into three sub-clusters: C1, C2, andC3. Results obtained using the chosen methodology do not show theexistence of circulating BCoV recombinant variants. Indeed, all of theBCoVs in our study are of non-recombinant genotype C2C2C2 (seeTable 2). All of the sequences of the 24 BCoVs and BCoVs-like comingfrom the US and included in the phylogenetic analysis are of a non-recombinant C1C1C1 genotype. In the same way, the sequences ofthree BCoV prototypes and the CrCoV included in the study are of anon-recombinant C3C3C3 genotype. Within this analysis of putativerecombinant detection, only the coronavirus strain HEC 4408 shows arecombinant genotype of type C3C2C3. It is worthwhile to highlightthat the HEC 4408 finds his root among the French isolates of BCoV(Fig. 2) and thus represents an independent sub-group within theBCoV cluster. The HEC 4408, serologically distinct from HCoV-OC43,illustrates the capacity of BCoV for spill-over independently of its rolein the emergence of HCoV-OC43.

In the end, these results suggest that HCoV-OC43 and BCoV do notseem to evolve along the same dynamics, as it points to a generationof recombinant variants that seems very active in HCoV-OC43 ascompared to BCoV. Given that these two viruses are extremely closewith respect to genetic and epidemiological characteristics, thisdifference could be linked to different environmental constraints(sensu-lato). In this light, one may conceive of a lesser genetic diversityof the animal host (animal livestock versus human) resulting fromdomestication and breeding practices by human, and a lesser geographicmobility of animals, thereby weakening the BCoV diversifying selectionprocess and decreasing potential co-infection and thus recombinationevents. These hypotheses must however be tested on a greater viralgenetic dataset.

The results involving DcCoV sequences available in GenBank show apotential genotype C2 « COUTLIER » C2. The potential outlier topology

Table 2Distribution of sequences of BCoV and BCoV-like among the sub-clusters on nsp12, S, andN tree.

nsp12 S N Conclusion

14 BCoVs from France (this study) C2 C2 C2 C2C2C2Three Prototype strains⁎ and one CrCoV C3 C3 C3 C3C3C312 BCoV and 12 BCoV-like from USA C1 C1 C1 C1C1C1HEC 4408 C3 C2 C3 C3C2C3Three DcCoV C2 COUTLIER C2 C2COUTLIERC2

⁎ BCoV Mebus, Kakegawa and Quebec.

describes the fact that, in the S gene tree, the DcCoV sequences form apotential third cluster that is distinct from that of BCoV andHCoV-OC43.

The complementary phylogenetic analysis involving a great numberof S gene sequences presented on Fig. S4 (supplementary material)highlights three clusters that group each of the HCoV-OC43s, BCoVsand BCoVs-like, and DcCoVs, respectively. In the cluster of BCoV andBCoV-like strains, a geographic distribution may be noted, having a so-called European sub-cluster and a so-called America-Asia sub-cluster.This distribution suggests that the divergence of the two sub-clustersof BCoVs is a relatively recent phenomenon with regards to the historyof livestock cattle. Indeed, the Bos taurus taurus livestock cattle of theOldWorld (Europe, Asia, and Africa) are issued from the domesticationfacilities of the aurochs, a wild species living exclusively in the OldWorld. This domestication event occurred around 10,000 years ago.Livestock cattle arrived on the North and South American continentafter the discovery of the New World in the XIth century (Bollonginoet al., 2012; Loftus et al., 1994). The distinction of genetic clusters corre-sponding to the BCoV of different geographic origins could be attributedto the diffusion of strains favoring the exchange of animals incurred byinternational commerce. In Europe, France is the largest producer andexporter of cattle. Moreover, France exports live cattle for reproductionto Italy, Spain, Greece, and Germany, and also to Algeria, Tunisia, andLibya. The import–export of fresh and frozen beef between the US andthe European Union has been minimal, especially after 1998 at whichpoint the EU appealed to the World Trade Organization (WTO) to pro-hibit the import of beef for purposes of food safety. The final decisionof the WTO was favorable to the European Union, but was not handeddown until 2007. However, between 1998 and 2007, commercebetween US and EU was greatly weakened.

The United States exports beef to Asia, especially to Japan and toSouth Korea.We recall that the BCoVs, aswell as the BCoVs-like collectedfrom ruminants in the USA and in South Korea are very similar. It is alsoworthwhile to note that the BCoV is not included in the WTO list ofillness being monitored in international exchange.

In conclusion, the present epidemiological study has made it possi-ble for the first time to obtain partial sequences of BCoV originatingfrom France. The results obtained, despite their limits in methodologyand in the number of sequences, suggest the existence of an evolutivepattern by recombination that is different between two very similarhuman and bovine coronaviruses. This study also draws light to thescarce availability of BCoV sequences and genetic analyses despite thefact that this pathogenic agent was identified over 50 years ago andthat it seems to have a significant sanitary impact on livestock cattle.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.meegid.2016.03.006.

Funding

This work was supported by the French Agence Nationale de laRecherche (ANR) on the behalf of EPICOREMproject (Eco-Epidemiologyof Coronaviruses, from Wildlife to Human: Emergence Threat Assess-ment), grant ANR-13-BSV3-0013.

Acknowledgements

We are grateful to Stephane Pronost of departmental laboratoryLABEO Franck Duncombe of Calvados and to Victor Carpinshi, FabienneBenoît, and Delphine Esperet of departmental laboratory of Manche, forproviding us the field samples. We thank all of our partners of theEPICOREM Consortium.

References

Adams, M.J., Carstens, E.B., 2012. Ratification vote on taxonomic proposals to the Interna-tional Committee on Taxonomy of Viruses (2012). Arch. Virol. 157, 1411–1422.

Aldrich, J., 1997. Others RA Fisher and the making of maximum likelihood 1912–1922.Stat. Sci. 12, 162–176.

191N. KIN et al. / Infection, Genetics and Evolution 40 (2016) 186–191

Alekseev, K.P., Vlasova, A.N., Jung, K., Hasoksuz, M., Zhang, X., Halpin, R., Wang, S., Ghedin,E., Spiro, D., Saif, L.J., 2008. Bovine-like coronaviruses isolated from four species ofcaptive wild ruminants are homologous to bovine coronaviruses, based on completegenomic sequences. J. Virol. 82, 12422–12431.

Beaudeau, F., Björkman, C., Alenius, S., Frössling, J., 2010. Spatial patterns of bovine CoronaVirus and bovine respiratory syncytial virus in the Swedish beef cattle population.Acta Vet. Scand. 52, 33.

Bollongino, R., Burger, J., Powell, A., Mashkour, M., Vigne, J.-D., Thomas, M.G., 2012.Modern taurine cattle descended from small number of near-Eastern Founders.Mol. Biol. Evol. 29, 2101–2104.

Cavanagh, D., Davis, P.J., Cook, J.K.A., 1992. Infectious bronchitis virus: evidence for recom-bination within the Massachusetts serotype. Avian Pathol. 21, 401–408.

Cebra, C.K., Mattson, D.E., Baker, R.J., Sonn, R.J., Dearing, P.L., 2003. Potential pathogens infeces from unweaned llamas and alpacas with diarrhea. J. Am. Vet. Med. Assoc. 223,1806–1808.

Chouljenko, V.N., Lin, X.Q., Storz, J., Kousoulas, K.G., Gorbalenya, A.E., 2001. Comparison ofgenomic and predicted amino acid sequences of respiratory and enteric bovinecoronaviruses isolated from the same animal with fatal shipping pneumonia. J. Gen.Virol. 82, 2927–2933.

Chung, J.-Y., Kim, H.-R., Bae, Y.-C., Lee, O.-S., Oem, J.-K., 2011. Detection and characteriza-tion of bovine-like coronaviruses from four species of zoo ruminants. Vet. Microbiol.148, 396–401.

Decaro, N., Campolo, M., Mari, V., Desario, C., Colaianni, M.L., Di Trani, L., Cordioli, P.,Buonavoglia, C., 2009a. A candidate modified-live bovine coronavirus vaccine: safetyand immunogenicity evaluation. New Microbiol. 32, 109–113.

Decaro, N., Mari, V., Campolo, M., Lorusso, A., Camero, M., Elia, G., Martella, V., Cordioli, P.,Enjuanes, L., Buonavoglia, C., 2009b. Recombinant canine coronaviruses related totransmissible gastroenteritis virus of swine are circulating in dogs. J. Virol. 83,1532–1537.

Erles, K., Toomey, C., Brooks, H.W., Brownlie, J., 2003. Detection of a group 2 coronavirusin dogs with canine infectious respiratory disease. Virology 310, 216–223.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap.Evolution 39, 783.

Gélinas, A.-M., Boutin, M., Sasseville, A., Dea, S., 2001. Bovine coronaviruses associatedwith enteric and respiratory diseases in Canadian dairy cattle display differentreactivities to b i N antib/i N −HE monoclonal antibodies and distinct amino acidchanges in their HE, S and ns4. 9 protein. Virus Res. 76, 43–57.

Gorbalenya, A.E., Enjuanes, L., Ziebuhr, J., Snijder, E.J., 2006. Nidovirales: evolving thelargest RNA virus genome. Virus Res. 117, 17–37.

Hasoksuz, M., Sreevatsan, S., Cho, K.-O., Hoet, A.E., Saif, L.J., 2002. Molecular analysis of theS1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirusisolates. Virus Res. 84, 101–109.

Hasoksuz, M., Alekseev, K., Vlasova, A., Zhang, X., Spiro, D., Halpin, R., Wang, S., Ghedin, E.,Saif, L.J., 2007. Biologic, antigenic, and full-length genomic characterization of abovine-like coronavirus isolated from a giraffe. J. Virol. 81, 4981–4990.

Hasoksuz, M., Vlasova, A., Saif, L.J., 2008. Detection of Group 2a Coronaviruses withEmphasis on Bovine and Wild Ruminant Strains. In: Cavanagh, D. (Ed.), SARS- andOther CoronavirusesMethods in Molecular Biology. Humana Press, pp. 43–59.

Herrewegh, A.A., Smeenk, I., Horzinek, M.C., Rottier, P.J., de Groot, R.J., 1998. Feline coro-navirus type II strains 79-1683 and 79-1146 originate from a double recombinationbetween feline coronavirus type I and canine coronavirus. J. Virol. 72, 4508–4514.

Institut National de Veille Sanitaire (INVS), 2015. Bulletin hebdomadaire internationalN°517, du 12 au 18 août.

Jeong, J.-H., Kim, G.-Y., Yoon, S.-S., Park, S.-J., Kim, Y.-J., Sung, C.-M., Shin, S.-S., Lee, B.-J.,Kang, M.-I., Park, N.-Y., Koh, H.-B., Cho, K.-O., 2005. Molecular analysis of S gene ofspike glycoprotein of winter dysentery bovine coronavirus circulated in Korea during2002–2003. Virus Res. 108, 207–212.

Kin, N., Miszczak, F., Lin, W., Gouilh, M.A., Vabret, A., 2015. Consortium, E. GenomicAnalysis of 15 Human Coronaviruses OC43 (HCoV-OC43s) Circulating in Francefrom 2001 to 2013 reveals a high intra-specific diversity with new recombinantgenotypes. Viruses 7, 2358–2377.

Lau, S., Lee, P., Tsang, A.K.L., Yip, C.C.Y., Tse, H., Lee, R.A., So, L.-Y., Lau, Y.-L., Chan, K.-H.,Woo, P.C.Y., Yuen, K.-Y., 2011. Molecular epidemiology of human coronavirus OC43reveals evolution of different genotypes over time and recent emergence of a novelgenotype due to natural recombination. J. Virol. 85, 11325–11337.

Lauber, C., Ziebuhr, J., Junglen, S., Drosten, C., Zirkel, F., Nga, P.T., Morita, K., Snijder, E.J.,Gorbalenya, A.E., 2012. Mesoniviridae: a proposed new family in the orderNidovirales formed by a single species of mosquito-borne viruses. Arch. Virol. 157,1623–1628.

Loftus, R.T., Machugh, D.E., Bradley, D.G., Sharp, P.M., Cunningham, P., 1994. Evidence fortwo independent domestications of cattle. Proc. Natl. Acad. Sci. 91, 2757–2761.

Majhdi, F., Minocha, H.C., Kapil, S., 1997. Isolation and characterization of a coronavirusfrom elk calves with diarrhea. J. Clin. Microbiol. 35, 2937–2942.

Martínez, N., Brandão, P.E., de Souza, S.P., Barrera, M., Santana, N., de Arce, H.D., Pérez, L.J.,2012. Molecular and phylogenetic analysis of bovine coronavirus based on the spikeglycoprotein gene. Infect. Genet. Evol. 12, 1870–1878.

McIntosh, K., Dees, J.H., Becker, W.B., Kapikian, A.Z., Chanock, R.M., 1967. Recovery intracheal organ cultures of novel viruses from patients with respiratory disease.Proc. Natl. Acad. Sci. U. S. A. 57, 933.

Mebus, C.A., Stair, E.L., Rhodes, M.B., Twiehaus, M.J., 1973. Pathology of neonatal calfdiarrhea induced by a coronavirus-like agent. Vet. Pathol. Online 10, 45–64.

Park, S.-J., Jeong, C., Yoon, S.-S., Choy, H.E., Saif, L.J., Park, S.-H., Kim, Y.-J., Jeong, J.-H., Park,S.-I., Kim, H.-H., Lee, B.-J., Cho, H.-S., Kim, S.-K., Kang, M.-I., Cho, K.-O., 2006. Detectionand characterization of bovine coronaviruses in fecal specimens of adult cattle withdiarrhea during the warmer seasons. J. Clin. Microbiol. 44, 3178–3188.

Park, S.J., Lim, G.K., Park, S.I., Kim, H.H., Koh, H.B., Cho, K.O., 2007. Detection and molecularcharacterization of calf diarrhoea bovine coronaviruses circulating in South Koreaduring 2004–2005. Zoonoses Public Health 54, 223–230.

Peiris, J.S.M., Guan, Y., Yuen, K.Y., 2004. Severe acute respiratory syndrome. Nat. Med. 10,S88–S97.

Robinson, T.P., Wint, G.R.W., Conchedda, G., Van Boeckel, T.P., Ercoli, V., Palamara, E.,Cinardi, G., D'Aietti, L., Hay, S.I., Gilbert, M., 2014. Mapping the global distribution oflivestock. PLoS One 9, e96084.

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructingphylogenetic trees. Mol. Biol. Evol. 4, 406–425.

Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide substitutions in thecontrol region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol.10, 512–526.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecularevolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.

Tsunemitsu, H., El-Kanawati, Z.R., Smith, D.R., Reed, H.H., Saif, L.J., 1995. Isolation ofcoronaviruses antigenically indistinguishable from bovine coronavirus from wildruminants with diarrhea. J. Clin. Microbiol. 33, 3264–3269.

Vijgen, L., Keyaerts, E., Moes, E., Thoelen, I., Wollants, E., Lemey, P., Vandamme, A.-M., VanRanst, M., 2005. Complete genomic sequence of human coronavirus OC43: molecularclock analysis suggests a relatively recent zoonotic coronavirus transmission event.J. Virol. 79, 1595–1604.

Woo, P.C.Y., Lau, S.K.P., Huang, Y., Yuen, K.-Y., 2009. Coronavirus diversity, phylogeny andinterspecies jumping. Exp. Biol. Med. 234, 1117–1127.

Woo, P.C.Y., Lau, S.K.P., Lam, C.S.F., Lau, C.C.Y., Tsang, A.K.L., Lau, J.H.N., Bai, R., Teng, J.L.L.,Tsang, C.C.C., Wang, M., Zheng, B.-J., Chan, K.-H., Yuen, K.-Y., 2012. Discovery of sevennovel mammalian and avian coronaviruses in the Genus deltacoronavirus supportsBat Coronaviruses as the Gene Source of alphacoronavirus and betacoronavirus andavian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.J. Virol. 86, 3995–4008.

Woo, P.C.Y., Lau, S.K.P., Wernery, U., Wong, E.Y.M., Tsang, A.K.L., Johnson, B., Yip, C.C.Y.,Lau, C.C.Y., Sivakumar, S., Cai, J.-P., Fan, R.Y.Y., Chan, K.-H., Mareena, R., Yuen, K.-Y.,2014. Novel betacoronavirus in dromedaries of the Middle East, 2013. Emerg. Infect.Dis. 20, 560–572.

Yoo, D., Pei, Y., 2001. Full-length genomic sequence of bovine coronavirus (31 kb).completion of the open reading frame 1a/1b sequences. Adv. Exp. Med. Biol. 494,73–76.

Zaki, A.M., van Boheemen, S., Bestebroer, T.M., Osterhaus, A.D.M.E., Fouchier, R.A.M., 2012.Isolation of a novel coronavirus from a manwith pneumonia in Saudi Arabia. N. Engl.J. Med. 367, 1814–1820.

Zhang, X.M., Kousoulas, K.G., Storz, J., 1991. Comparison of the nucleotide and deducedamino acid sequences of the S genes specified by virulent and avirulent strains ofbovine coronaviruses. Virology 183, 397–404.

Zhang, X.M., Herbst, W., Kousoulas, K.G., Storz, J., 1994. Biological and genetic characteri-zation of a hemagglutinating coronavirus isolated from a diarrhoeic child. J. Med.Virol. 44, 152–161.

Zhang, X., Hasoksuz, M., Spiro, D., Halpin, R., Wang, S., Vlasova, A., Janies, D., Jones, L.R.,Ghedin, E., Saif, L.J., 2007. Quasispecies of bovine enteric and respiratorycoronaviruses based on complete genome sequences and genetic changes after tissueculture adaptation. Virology 363, 1–10.